EP0580635B2 - Modified plant viruses as vectors - Google Patents

Modified plant viruses as vectors Download PDF

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EP0580635B2
EP0580635B2 EP92907583A EP92907583A EP0580635B2 EP 0580635 B2 EP0580635 B2 EP 0580635B2 EP 92907583 A EP92907583 A EP 92907583A EP 92907583 A EP92907583 A EP 92907583A EP 0580635 B2 EP0580635 B2 EP 0580635B2
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virus
plant
rna
modified
plants
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EP0580635A1 (en
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George Peter Lomonossoff
John Emil Johnson
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AXIS GENETICS PLC EN PURDUE RESEARCH FOUNDATION
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Purdue Research Foundation
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    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
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Definitions

  • This invention relates to the use of viruses as carriers (vectors) for the production or presentation of foreign peptides. More particularly, the invention relates to the genetic manipulation of viral nucleic acid by incorporation of foreign nucleic acid sequences which are expressed as peptides in the virus particle (virion).
  • virus particle virus particle
  • foreign as applied to a peptide or to the nucleic acid encoding therefor, signifies peptide or nucleic acid sequences which are not native to the plant virus used as a vector. Such sequences can be alternatively described as exogenous or heterologous sequences.
  • peptide includes small peptides and polypeptides.
  • viruses as carriers of foreign peptides has been extensively explored in the field of composite virus vaccines.
  • Such vaccines are based on chimeric viruses which are hybrids of different animal virus components.
  • the major component of such hybrids is derived from a virus which is or has been rendered harmless and the minor component is a selected antigenic component of a pathogenic virus.
  • a pox virus such as vaccinia or an attenuated poliovirus may be used as a vector for immunogenic components of other animal viruses including human viruses.
  • Such vaccines are produced from viruses grown in cell culture systems which are expensive to design and run.
  • the composite virus approach involves genetic manipulation of live, animal-infecting viruses, with the risk that mutations may give rise to novel forms of the virus with altered infectivity, antigenicity and/or pathogenicity.
  • the animal virus used as the vector is often a virus to which the animal may already have been exposed, and the animal may already be producing antibodies to the vector. The vector may therefore be destroyed by the immune system before the incorporated antigenic site of the second virus induces an immune response.
  • the present invention avoids the above-mentioned disadvantages by the use of a radically different type of virus component in the design of chimeric viruses expressing foreign sequences. Moreover, although the invention has particular relevance to the solution of problems encountered in the production of virus vaccines, it is much wider both in concept and field of application as indicated hereinafter.
  • the present invention utilises plant viruses as vector systems for the expression of foreign nucleotide sequences ie nucleotide sequences (RNA or DNA) which are not present in plant viruses, as found in Nature, and which in consequence code for peptides not normally found in any naturally occurring plant virus.
  • RNA or DNA foreign nucleotide sequences
  • the present invention provides assembled particles of a plant virus containing a predetermined foreign peptide as part of the coat protein of the virus, the particles being capable of replicating and assembling in plants, and having been assembled in whole plants or in plant cells, wherein the viral coat protein has a ⁇ -barrel structure, and the foreign peptide is inserted in a loop connecting ⁇ sheets of the viral coat protein.
  • modified viruses are therefore modified forms of the native viruses and for convenience will be referred to as modified viruses.
  • modified viruses may be formed from any biologically useful peptides (usually polypeptides) the function of which requires a particular conformation for its activity. This may be achieved by association of the peptide with a larger molecule eg to improve its stability or mode of presentation in a particular biological system.
  • peptide hormones are peptide hormones; enzymes; growth factors; antigens of protozoal, viral, bacterial, or fungal origin; antibodies including anti-idiotypic antibodies; immunoregulators and cytokines eg interferons and interleukins; receptors; adhesins; and parts or precursors of any of the foregoing types of peptide.
  • the peptide preferably contains more than 5 amino acids.
  • the antigenic peptides which are the basis of vaccines, particularly animal (including human) virus vaccines.
  • the invention provides an especially attractive epitope presentation system.
  • the antigenic peptide component will be sited appropriately on the virus particle so as to be easily recognised by the immune system, for example by location on an exposed part of the coat protein of the virus.
  • the present invention comprises assembled particles of a modified plant virus containing an antigen derived from a pathogen eg an animal virus, incorporated in an exposed surface of the coat protein of the plant virus.
  • the invention also comprises the use of such assembled modified plant virus particles as the immunogenic component of a vaccine.
  • the plant viral nucleic acid is modified by introducing a nucleotide sequence coding for the foreign peptide eg an animal virus antigen at that part of the plant viral genome which codes for an exposed portion of the coat protein, infecting plants or plant cells with the modified viral nucleic acid, and harvesting assembled particles of the modified virus.
  • This procedure is best carried out by direct manipulation of the DNA of the virus in the case of DNA viruses or by manipulation of a cDNA corresponding to the RNA of an RNA virus.
  • an RNA transcript of the modified DNA is usually prepared for inoculation of plant cells or preferably whole plants so as to achieve a multiplication stage prior to the harvesting of assembled particles of the modified virus.
  • the DNA itself is introduced into the plant.
  • the foreign peptide is initially expressed as part of the capsid protein and is thereby produced as part of the whole virus particle.
  • the peptide may thus be produced as a conjugate molecule intended for use as such.
  • the genetic modification of the virus may be designed in order to permit release of the desired peptide by the application of appropriate agents which will effect cleavage from the virus particle.
  • infective inoculant DNA or RNA transcript
  • an initial inoculant may be used to infect plants and the resulting modified virus may be passaged in the plants to produce whole virus or viral RNA as inoculant for subsequent batches.
  • the viruses which are used in the present invention are those whose coat proteins have a ⁇ -barrel structure.
  • An advantage of the use of viruses which have this structure is that the loops between the individual strands of ⁇ -sheet provide convenient sites for the insertion of foreign peptides. Modification of one or more loops is the strategy for the expression of foreign peptides in accordance with the present invention.
  • This group includes the comoviruses such as cowpea mosaic virus and bean pod mottle virus, and the nepoviruses such as tomato ringspot virus and strawberry latent ringspot virus.
  • An advantage of the comoviruses is that their capsid contains sixty copies each of 3 different ⁇ -barrels which can be individually manipulated.
  • Other virus groups with similar 3-dimensional structures but a single type of ⁇ -barrel include the tombusviruses and the sobemoviruses.
  • the foreign RNA or DNA may be inserted into the plant virus genome in a variety of configurations.
  • it may be inserted as an addition to the existing nucleic acid or as a substitution for part of the existing sequence, the choice being determined largely by the structure of the capsid protein and the ease with which additions or replacements can be made without interference with the capacity of the genetically-modified virus to assemble in plants. Determination of the permissible and most appropriate size of addition or deletion for the purposes of this invention may be achieved in each particular case by experiment in the light of the present disclosure. The use of addition inserts appears to offer more flexibility than replacement inserts in some instances.
  • the inserted heterologous nucleotide sequence may include those coding for amino acids which are readily cleaved so that, after the multiplication stage, the desired material may be separated from the virus particles.
  • the desired material may be separated from the virus particles.
  • epitopes which can be expressed on the surface of the CPMV capsids are those from picornaviruses such as foot-and-mouth disease virus (FMDV), poliovirus, human rhinovirus (HRV) and hepatitis A virus (HAV), epitopes associated with either gp41 or gp120 of human immunodeficiency virus (HIV) and the epitope derived from the major coat protein of human papillomavirus (HPV).
  • picornaviruses such as foot-and-mouth disease virus (FMDV), poliovirus, human rhinovirus (HRV) and hepatitis A virus (HAV)
  • epitopes associated with either gp41 or gp120 of human immunodeficiency virus (HIV) and the epitope derived from the major coat protein of human papillomavirus (HPV).
  • the present invention has many advantages over conventional vaccines, recombinant vaccines based on animal viruses, and peptide vaccines, for example:
  • CPMV plant virus cowpea mosaic comovirus
  • FMDV foot and mouth disease virus
  • HRV human rhinovirus
  • the third virus selected was human immune deficiency virus (HIV) which bears no similarity to any known plant virus, and for which no successful vaccines are currently available.
  • HIV human immune deficiency virus
  • one trapezoid represents one ⁇ -barrel.
  • Figure 2 depicts the secondary structure and connectivity of a canonical ⁇ -barrel.
  • the individual strands of ⁇ sheet are labelled B through G and the amino- (NH 2 ) and carboxy- (COOH) termini of the protein are indicated.
  • Figure 3 depicts the plasmids (A) pPMM2902 and (B) pBT7-123.
  • the stippled regions represent the CPMV-specific regions of the plasmids with the coding regions being indicated by the wider portions on which the various virus-encoded proteins are marked. Relevant restriction enzyme sites are indicated. Details of the construction of the plasmids are given in Holness et al (1989) and Dessens and Lomonossoff (1991).
  • Figure 4 depicts the region of CPMV M RNA which encodes the amino-terminal 40 amino acids of VP23.
  • the numbers below the nucleotide sequence refer to the M RNA sequence and the position of the unique Nhel site is indicated.
  • the amino acids involved in forming the ⁇ B and ⁇ C strands of VP23 are indicated above the amino acid sequence of the protein which is shown using the standard one-letter code.
  • Figure 5 depicts (A) the nucleotide sequence of the oligonucleotides used in the construction of pFMDV together with the amino acid sequence encoded by the top (positive) strand and (B) the structure of VP23 after insertion of the FMDV-specific oligonucleotides.
  • the arrowed region indicates the extent of the inserted FMDV epitope.
  • the Nhel site not restored during the cloning is indicated by xNhel.
  • the diagnostic Bgl11 site present in the inserted sequence is also indicated.
  • Figure 6 depicts the construction of plasmid pFMDV.
  • the representation of the various CPMV-specific regions is as in Figure 3.
  • the FMDV-specific region which is inserted into VP23 is shown as the black segment in the CPMV-specific coding region.
  • Figure 7 depicts plasmid pMT7-601. The representation of the various CPMV-specific regions is as in Figure 3. Relevant restriction sites are indicated.
  • Figure 8 depicts the construction of a "substitution" vector by site-directed mutagenesis, The asterisk indicates the T residue that is changed to a C by site-directed mutagenesis, thereby creating a novel Aat11 site.
  • Figure 9 depicts (A) the nucleotide sequence of the oligonucleotides used in the construction of pMT7-HIV together with the amino acid sequence encoded by the top (positive) strand and (B) the structure of VP23 after insertion of the HIV-specific oligonucleotides.
  • the arrowed region indicates the extent of the inserted HIV epitope.
  • the diagnostic Pvu1 site present in the inserted sequence is also indicated.
  • Figure 10 depicts (A) the nucleotide sequence of the oligonucleotides used in the construction of pMT7-HRV together with the amino acid sequence encoded by the top (positive) strand and (B) the structure of VP23 after insertion of the HRV-specific oligonucleotides.
  • the arrowed region indicates the extent of the inserted HRV epitope.
  • the diagnostic Clal site present in the inserted sequence is also indicated.
  • Figure 11 depicts the construction of plasmids pMT7-HIV and pMT7-HRV.
  • the representation of the various CPMV-specific regions is as in Figure 3.
  • the HIV-and HRV-specific regions inserted into VP23 are shown as the black segment in the CPMV-specific coding region.
  • Figure 12 depicts a "Western blot" of protein extracts of the leaves of five cowpea plants (lanes A to E) inoculated with a mixture of pMT7-FMDV-1 and PBT7-123 transcripts. The blot was probed with serum specific for the FMDV epitope. Lanes “mock” and “wt” contain extracts of leaves which were either mock-inoculated or inoculated with wild-type CPMV RNA, respectively. The lane marked "virus” contains purified wild-type CPMV. The sizes of marker proteins are shown on the right-hand side of the blot.
  • Comoviruses are a group of at least fourteen plant viruses which predominantly infect legumes. Their genomes consist of two molecules of single-stranded, positive-sense RNA of different sizes which are separately encapsidated in isometric particles of approximately 28nm diameter.
  • the two types of nucleoprotein particles are termed middle (M) and bottom (B) component as a consequence of their behaviour in caesium chloride density gradients, the RNAs within the particles being known as M and B RNA, respectively
  • Both types of particle have an identical protein composition, consisting of 60 copies each of a large (VP37) and a small (VP23) coat protein.
  • comovirus preparations contain a variable amount of empty (protein-only) capsids which are known as top (T) component.
  • cowpea mosaic virus CPMV
  • M and B RNA are polyadenylated and have a small protein (VPg) covalently linked to their 5'terminus.
  • VPg small protein
  • Both RNAs from CPMV have been sequenced and shown to consist of 3481 (M) and 5889 (B) nucleotides, excluding the poly (A) tails (van Wezenbeek et al. 1983; Lomonossoff and Shanks, 1983).
  • RNAs contain a single, long open reading frame, expression of the viral gene products occurring through the synthesis and subsequent cleavage of large precursor polypeptides. Though both RNAs are required for infection of whole plants, the larger B RNA is capable of independent replication in protoplasts, though no virus particles are produced in this case (Goldbach et al., 1980). This observation, coupled with earlier genetic studies, established that the coat proteins are encoded by M RNA.
  • the capsids of these latter viruses are composed of 180 identical coat protein subunits, each consisting of a single ⁇ -barrel domain. These can occupy three different positions, A, B and C, within the virions ( Figure 1).
  • the two coat proteins of CPMV were shown to consist of three distinct ⁇ -barrel domains, two being derived from VP37 and one from VP23.
  • each CPMV particle is made up of 180 ⁇ -barrel structures.
  • the single domain from VP23 occupies a position analogous to that of the A type subunits of TBSV and SBMV, whereas, the N-and C-terminal domains of VP37 occupy the positions of the C and B type subunits respectively ( Figure 1).
  • BPMV bean pod mottle virus
  • each ⁇ -barrel consists principally of 8 strands of antiparallel ⁇ -sheet connected by loops of varying length.
  • the connectivity and nomenclature of the strands is given in Figure 2.
  • the flat ⁇ -sheets are named the B,C,D,E,F,G,H and I sheets, and the connecting loops are referred to as the ⁇ B- ⁇ C, ⁇ D- ⁇ E, ⁇ F- ⁇ G and ⁇ H- ⁇ I loops.
  • the comoviruses are also structurally related to the animal picornaviruses.
  • the capsids of picornaviruses consist of 60 copies of each of three different coat proteins VP1, VP2 and VP3 each one consisting of a single ⁇ -barrel domain. As in the case of comoviruses, these coat proteins are released by cleavage of a precursor polyprotein and are synthesised in the order VP2 - VP3 - VP1.
  • Comparison of the 3-dimensional structure of CPMV with that of picomaviruses has shown that the N- and C- terminal domains of VP37 are equivalent to VP2 and VP3 respectively and that VP23 are equivalent to VP1 ( Figure 1).
  • the equivalence between structural position and gene order suggests that VP37 corresponds to an uncleaved form of the two picornavirus capsid proteins, VP2 and VP3.
  • B RNA purified from virions to provide the proteins required for viral replication with pPMM2902 might be cross-contaminated with wild-type M RNA
  • pBT7-123 a full-length cDNA clone of B RNA
  • the full-length copy of B RNA is immediately downstream of a modified T7 promoter.
  • transcripts identical in size to natural B RNA can be synthesised by T7 RNA polymerase.
  • a mixture of transcripts from pPMM2902 and pBT7-123 gives rise to a full virus infection when electroporated into cowpea protoplasts, and therefore replaces the use of natural B RNA.
  • FMDV picornavirus foot and mouth disease
  • HRV human rhinovirus
  • HAV lentiretrovirus human immune deficiency virus
  • the positive sense oligonucleotide contains the sequence encoding amino acid residues 136-160 from VP1 of FMDV serotype O 1 strain BFS 1860.
  • the nucleotide sequence of oligonucleotides was designed to take account of the codon usage preference found in CPMV and includes a BgI11 site in the middle of the sequence to facilitate screening. When annealed, the oligonucleotides give a double-stranded DNA sequence with Nhel-compatible ends.
  • the oligonucleotides can be inserted into the unique Nhel site of pPMM2902.
  • the effect of such an insertion on the sequence of the VP23 is shown in Figure 5B.
  • the FMDV-specific oligonucleotides were initially ligated into an M13 subclone of pPMM2902 which contained the sequence encoding VP23. This was done to enable clones harbouring the FMDV-specific sequence to readily be identified by sequence analysis. All the standard DNA manipulations were carried out according to Maniatis et al (1982). Details of the construction of pFMDV are given below and are shown diagrammatically in Figure 6.
  • the plasmid pPMM2902 was digested with the restriction enzyme Sstl which cuts twice within the CPMV M RNA-specific sequence at positions 2296 and 3423 but does not cut within the sequence of the plasmid pPM1. Following agarose gel electrophoresis, and both the large (6.0kb) the small (1.1 kb) fragment were purified by electro-elution from the gel. The 1.1 kb Sstl fragment was ligated into the Sst1-cut, phosphatase-treated replicative form DNA from the bacteriophage M13mp18. The ligation mixture was used to transform E.coli strain JM101 using the calcium chloride procedure. Plaques containing the 1.1 kb Sstl fragment from M RNA were identified by the Lac complementation assay and DNA sequence analysis and one, M13-JR1 was selected for further work.
  • Sstl restriction enzyme
  • the annealed oligonucleotides were ligated into Nhe-1-digested M13-JR1, the ligation mixture used to transform E. coli strain JM101 and the transformation mixture plated out on a lawn of JM101. A large number of plaques were found on the plates, 20 of which were selected for sequence analysis. Bacteriophage were propagated in JM101 and the single-stranded DNA isolated exactly as described by Sanger et al (1980).
  • the nucleotide sequence of the region of the bacteriophage DNA around the Nhel site was determined by the dideoxy method as modified by Biggin et al (1983), using an 18mer,5' AGT-TAC-TGC-TGT-AAC-GTC-3', complementary to nucleotides 2735-2752 of the M RNA sequence, as primer. Of the plaques analysed, one, designated M13-ushal, had a single copy of the desired sequence in the correct orientation.
  • pFMDV plasmid DNA was isolated by the method of Birnboim and Doly (1979) and further purified by centrifugation using caesium chloride/ethidium bromide gradients (Maniatis et al (1982).
  • spermidine hydrochloride 0.5mM each of UTP, ATP and CTP, 0.025mM GTP, 0.5mM GpppG, 0.05mg/ml BSA, 10mM DTT, 200 units/ml RNAguard and transcription was initiated by the addition of T7 RNA polymerase to a final concentration of 1400 units/ml. Incubation was at 37C for 2 hours. At 30, 60 and 90 minutes portions (5 ⁇ l per 1 ml transcription reaction) of a 5mM solution of GTP were added. Following transcription, EDTA was added to 15mM final concentration and the integrity of the transcripts was checked by electrophoresis on formaldehyde-containing agarose gels.
  • the transcription mixtures were extracted with 2 volumes of phenol/chloroform (1.1 v/v) and the nucleic acids precipitated twice with ethanol.
  • the nucleic acids were harvested by centrifugation, washed with ethanol and dried under vacuum.
  • the nucleic acids were dissolved in 50mM Tris-phosphate, pH 8.0 for inoculation on to plants.
  • the primary leaves of 10 day-old cowpea ( Vigna unguiculata var. California blackeye) were dusted with carborundum and a 1:1 (w/w) mixture of transcripts derived from pMT7-601 and pBT7-123 were applied to the leaves with gently rubbing.
  • a variety of transcript concentrations were used but in all cases the final inoculum volume was 50 ⁇ l.
  • the results obtained showed that when a total of 5 ⁇ g of each transcript was applied per primary leaf, 100% of plants inoculated routinely developed symptoms characteristic of a CPMV infection.
  • the presence of CPMV-specific sequences in both the inoculated and upper leaves of such plants was confirmed by "Dot blot" analysis.
  • Samples of the inoculated and trifoliate leaves were taken using a number 10 cork borer and macerated and extracted with 0.4 mls of 10mM sodium phosphate.
  • the samples were centrifuged and 5 ⁇ l of the supernatant was applied to nitrocellulose filters prewetted with 20XSSC.
  • the nucleic acid were cross-linked to the membranes by irradiation with u.v. light and probed for M RNA-specific sequences using a 32 P "oligo-labelled" (Feinberg and Vogelstein, 1983) probe consisting of nucleotides 482-2211 of the M RNA sequence.
  • the conditions for hybridisation and washing of the filters were as described by Maniatis et al (1982). After drying, the filters were autoradiographed. A strong hybridisation signal indicated the presence of CPMV-specific sequences.
  • pMT7-FMDV-I pMT7-601 and pFMDV were both digested with restriction enzyme Sstl, the digest from pMT7-601 being subsequently treated with calf intestinal phosphatase.
  • Sst1 cuts each plasmid twice at positions 2296 and 3423 of the M RNA-specific region to release a 1.1kb fragment. As discussed previously this Sstl fragment contains the region of VP23 encompassing the ⁇ B- ⁇ C loop where the FMDV loop insertion has been made.
  • the 1.1kb fragment from pFMDV and the 5.1kb fragment, encompassing the vector sequence and all the rest of the M RNA specific sequence, from pMT7-601 were recovered by electo-elution.
  • the two Sstl fragments were ligated together and the mixture transformed in E. coli strain JM83.
  • a number of carbenicillin-resistant colonies were picked, "minipreps" made and the plasmid DNA examined by restriction enzyme digests to identify recombinants containing the FMDV loop.
  • One such clone was identified, designated pMT7-FMDV-I and grown up on a large scale. All the DNA manipulations were as described for the construction of pFMDV and pMT7-601.
  • the creation of the Aat11 site enables the nucleotide sequence encoding the six amino acids from the native ⁇ B. ⁇ C loop in CPMV to be removed by digestion with Nhel and Aat11. The sequence can then be replaced by any sequence with Nhel- and Aat11-compatible ends.
  • the first sequence to be substituted into VP23 consisted of oligonucleotides encoding residues 735-752 from the transmembrane glycoprotein gp41 from human immunodeficiency virus (HIV-1). This sequence was selected as a synthetic peptide for this region is recognised in enzyme-linked immunosorbent assays (ELISA) by antisera from seropositive AIDS patients and is capable of inducing antibodies which neutralise a range of HIV-1 isolates (Kennedy et al , 1986; Chanh et al , 1986: Dagleish et al , 1988).
  • ELISA enzyme-linked immunosorbent assays
  • the second sequence consists of the nucleotide sequence encoding residues 85-99 from VP1 of human rhinovirus 14 (HRV14).
  • HRV14 human rhinovirus 14
  • the oligonucleotides were designed to contain restriction enzyme sites to facilitate screening.
  • the sequences of the oligonucleotides and the effect of the substitutions on the amino acid sequence of VP23 are shown in Figures 9 and 10.
  • the steps in the construction of pMT7-HIV and pMT7-HRV are given below and are shown diagrammatically in Figure 11.
  • M13-JR-1 (see Figure 6) was propagated in E. coli strain CJ236 and dU-containing single-stranded DNA isolated as described by Kunkel (1985).
  • the T to C mutation at position 2740 of the M RNA sequence was made by oligonucleotide-directed mutagenesis of dU-containing single-stranded M13-JR1 DNA using the primer CTG-CTG-TGA-CGT-CTG-AAA-A as described by Kunkel (1985). This resulted in the construction of clone M13-JRAat11.
  • the mutation was confirmed by dideoxy sequence analysis of single-stranded DNA (Biggin et al, 1983) and by restriction enzyme digestion of the double-stranded replication form DNA.
  • M13 clones harbouring the inserted sequences were identified by sequence analysis of the single-stranded bacteriophage DNA exactly as described previously for pFMDV Two clones, M13-HIV and M13-HRV containing the required sequences were identified and the double-stranded replicative form DNA was isolated shown to give the expected pattern of fragments on restriction enzyme digestion.
  • pMT7-HIV and pMT7-HRV were linearised by digestion with EcoR1. Transcription using T7 RNA polymerase was carried out exactly as described for pMT7-601 and pBT7-123. The resulting transcripts were identical in size to natural virion RNA.
  • transcripts capped with GpppG were prepared as previously described. 6 groups, each consisting of 5, 10 day old, cowpeas, were inoculated with the transcripts using the method previously described. In each case, the amount of transcript refers to the amount applied to an individual leaf. Group 1. Mock-inoculated with 50mM Tris-phosphate, pH 8.0 Group 2. Inoculated with 1.5 ⁇ g of natural CPMV virion RNA Group 3. Inoculated with 5 ⁇ g each of pMT7-601 + pBT7-123 transcripts Group 4.
  • pMT7-FMDV-I To demonstrate that modified viral capsid proteins were synthesised in the inoculated leaves of the group 4 plants, samples of the frozen leaf tissue were finely ground and extracted with 1 X Laemmli sample buffer. The extracts were electrophoresed on 15% polyacrylamide-SDS gels and the proteins transferred to nitrocellulose membranes using a Biorad semi-dry transfer cell. The membranes were probed either with serum raised against whole CPMV virus particles or with a serum raised against the synthetic oligopeptide, VPNLRGDLQVLAQKVARTLP(CG), corresponding to residues 141-160 of VP1 of FMDV strain O 1 .
  • pMT7-HIV As discussed above, the "dot blot" analysis of both the inoculated and systemic leaves from the Group 5 plants indicated that transcripts from pMT7-HIV can multiply and spread in whole plants. The levels of signal obtained and the fact that the infection went systemic show that the progeny RNA is encapsidated. To prove that the HI V-specific insert was retained in the progeny RNA, "dot blots" of extracts from the group 5 plants were probed with a HIV-insert specific probe. This was made by "oligo-labelling" the positive sense oligonucleotide used in the construction of pMT7-HIV (see Figure 9). The results obtained showed the presence of the HIV sequence in extracts of the inoculated leaves of the four plants which showed symptoms.
  • transcripts derived from pMT7-FMDV-I Five groups of five cowpea plants were inoculated with capped transcripts, prepared as previously described, as follows: Group 1 Mock-inoculated with 50mM Tris-phosphate, pH 8.0 Group 2 Inoculated with 0,5 ⁇ g of natural CPMV virion RNA Group 3 Inoculated with 5 ⁇ g GpppG capped pMT7-601 + pBT7-123 transcripts Group 4 Inoculated with 5 ⁇ g GpppG capped pMT7-FMDV-I + pBT7-123 transcripts
  • Symptoms were scored on a daily basis. 13 days post-inoculation triplicate leaf disk samples were taken from one inoculated and one trifoliate leaf of each plant. The samples were treated as follows:
  • Sample 1 Homogenised in 0.4mls 10mM sodium phosphate buffer, pH7.0, centrifuged and the supernatant recovered.
  • RNA extract Frozen in liquid nitrogen, finely ground and the nucleic acids extracted with phenol/chloroform. After ethanol precipitation, the nucleic acids were finally resuspended in 0.1 mls of water
  • Sample 3 Frozen in liquid nitrogen, finely ground and the powder dissolved in 0.1 ml 1X Laemmli sample buffer.
  • Dot blots were prepared from 5 ⁇ l aliquots of samples 1 and 2 and were probed with either a probe specific for nucleotides 482-2211 of CPMV M RNA (CPMV-specific probe), prepared as described previously, or with a probe specific for FMDV-specific insert. The latter was prepared by "oligo-labelling" the positive sense oligonucleotide shown in Figure 5.
  • Western blots were prepared from aliquots of sample 3 and probed for FMDV-specific epitopes as described previously ISEM was carried out on aliquots of sample 1.
  • ISEM using grids coated with anti-CPMV serum revealed the presence of copious numbers of CPMV particles in crude homogenates from both the inoculated and trifoliate leaves.
  • Western blot analysis of the protein extracts showed an absence of any FMDV epitopes.
  • the resulting pellet was resuspended overnight at 4°C in 3.5mls of 0.1M sodium phosphate, pH 7.0 and subsequently centrifuged in an Eppendorf centrifuge for 10 minutes. The supernatant was taken and made up to 4 mls with 0.1 M sodium phosphate pH 7.0 and 1ml of a solution containing 1 M NaCI, 20% PEG 6000 was added and the mixture incubated for 2 hours at room temperature. The resulting precipitate was collected by centrifugation in an Eppendorf centrifuge for 10 minutes, resuspended in 0.25mls of 10mM sodium phosphate, pH 7.0 and the solution clarified by re-centrifugation in an Eppendorf centrifuge for 10 minutes.
  • the supernatant which contains the virus particles, was then removed and stored at 4°C. It was estimated spectrophometrically that the virus concentration in the final suspension was approximately 1.5 mg/ml.
  • Western blot analysis of the virus using FMDV-specific antiserum revealed the presence of FMDV antigen associated with the small coat protein subunit of the chimaeric virus particles.
  • RNA extracted from transcript-inoculated leaves was passaged in plants. 5 ⁇ l samples of the RNA extract from a pMT7-FMDV-l-inoculated leaf were diluted to 50 ⁇ l with Tris-phosphate pH 8.0 and were inoculated on to the primary leaves of a batch of 5 cowpea plants. All the plants developed symptoms typical of a CPMV infection and at 23 days PI the primary leaves from the plants were harvested. The leaves were homogenised in 0.1M sodium phosphate buffer and virus extracted as described above except that the initial high speed pelleting step was omitted.
  • RNA extracted from the virus particles was of the expected size for M and B RNA of CPMV This demonstrates that chimaeric virus can be produced by passaging the RNA derived from transcript-inoculated leaves.
  • An experimental vaccine was prepared from virus preparation P1 by dispersion in sterile phosphate buffered saline (PBS) at a final concentration of 1 mg/ml. Guinea pigs were injected with 40 ⁇ g of the P1 vaccine on days 0 and 28. Preliminary results indicate that the animals produce antibodies against the FMDV loop, a response not seen when wild-type virus is injected.
  • PBS sterile phosphate buffered saline

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DK0580635T3 (da) 1998-01-19
DE69220485D1 (de) 1997-07-24
GR3035521T3 (en) 2001-06-29
ES2106173T3 (es) 1997-11-01
AR248426A1 (es) 1995-08-18
ZA922604B (en) 1992-12-30
HU9302954D0 (en) 1994-01-28
HUT65554A (en) 1994-06-28
DE69220485T2 (de) 1997-10-09
JPH06506583A (ja) 1994-07-28
EP0580635A1 (en) 1994-02-02
GB9108386D0 (en) 1991-06-05
EP0580635B1 (en) 1997-06-18
HU218010B (hu) 2000-05-28
JP3236614B2 (ja) 2001-12-10
AU661479B2 (en) 1995-07-27
NZ242430A (en) 1993-06-25
US5874087A (en) 1999-02-23
ATE154635T1 (de) 1997-07-15
US6884623B1 (en) 2005-04-26
ES2106173T5 (es) 2001-08-01
DK0580635T4 (da) 2001-03-19
WO1992018618A1 (en) 1992-10-29
DE69220485T3 (de) 2001-06-07
US7208655B1 (en) 2007-04-24

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